The proposed Microbicide Innovation Program fosters the development of new model systems (dual chamber and murine) that have the potential to substantially advance microbicide science. This approach is designed to improve methods for assessment of microbicide safety. The optimal microbicide should protect against infection without disrupting the mucosal environment or its mediators of host defense. The clinical trial failures with nonoxynol-9 and cellulose sulfate highlight the challenges in microbicide research and the need to establish better markers predictive of microbicide safety. The proposed studies address this gap. The primary objective of the R21 component is to establish two synergistic models of microbicide safety: an in vitro dual chamber model using primary human cervical epithelial cells and a murine model. Preliminary findings with these models demonstrate that the models would have predicted the increase in HIV acquisition observed in recently completed clinical trials. The microbicides disrupt the epithelium in vitro, as evidenced by a loss in transepithelial electrical resistance and in structural proteins and these changes are associated with an increased migration of cell-free HIV across the epithelium. In parallel studies, the drugs also trigger substantial changes in genital tract tissue architecture in mice following repeated vaginal application and the observed changes are associated with an increased susceptibility to genital herpes infection. Establishment of these two complementary models will contribute to efficient assessment of microbicide safety. During the R33 phase, both models will be translated into the preclinical pipeline by evaluating leading microbicide candidates, singly and in combination. Candidate microbicides will be introduced in the presence of cervicovaginal secretions and challenged in vitro with virus introduced in semen. The migration of both cell-free and cell-associated HIV will be tested in the dual chamber model system. In addition, during the R33 phase, the in vitro model will be expanded to assess the impact of microbicides on cells derived from women with human papillomavirus (HPV) associated dysplasia. While it is critical to assess the effect of microbicides on healthy genital tract cells and mucosa, it is highly likely that many women who choose to use a microbicide will be infected with a sexually transmitted infection. HPV is the most common sexually transmitted infection worldwide and changes in genital tract epithelium in response to microbicides may differ in women with HPV. These results will provide critical new data on microbicide safety in women with a sexually transmitted infection. Biomarkers predictive of microbicide safety are urgently needed. Tissue and murine models may provide more efficient strategies to assess microbicide safety by expanding existing models to include testing of primary cells. Development of an effective dual chamber and murine model may prove to be important in determining which candidate microbicides to move forward in the development pipeline. In addition, these models may provide a means to test the safety of microbicides in healthy women as well as those with underlying STIs.